Neurogenesis from Sox2 expressing cells in the adult cerebellar cortex

Abstract

We identified a rare undifferentiated cell population that is intermingled with the Bergmann glia of the adult murine cerebellar cortex, expresses the stem cell markers Sox2 and Nestin, and lacks markers of glial or neuronal differentiation. Interestingly, such Sox2⁺ S100^- cells of the adult cerebellum expanded after adequate physiological stimuli in mice (exercise), and Sox2⁺ precursors acquired positivity for the neuronal marker NeuN over time and integrated into cellular networks. In human patients, SOX2⁺ S100^- cells similarly increased in number after relevant pathological insults (infarcts), suggesting a similar expansion of cells that lack terminal glial differentiation.

Figure 1

Presence and expansion of undifferentiated Sox2⁺ cells in the cortex of the adult murine cerebellum. Within the adult cerebellar cortex, Sox2 is expressed in small cells within the Purkinje cell layer (PCL) that were believed to represent the Bergmann glia (a and arrows in b). While most of the Sox2 expressing cells within the PCL stained positive for the glial marker S100B (arrowheads in c–h), some of them exclusively expressed Sox2 (arrows in c–e). This population was further characterized by Nestin expression, as detected by Nestin-dependent GFP expression (arrows in f–h). Cre as nuclear surrogate for GFAP (arrowheads in i–k) and Blbp (arrowheads in l–n) mark Bergmann glia cells, but Sox2⁺  cells were consistently found that do not co-label with another (single) marker, of differentiation (arrows in i–n). Characterization of Sox2-expressing cells within the PCL was done by additional staining for specific cerebellar cell types. Sox2⁺ cells do not express markers of oligodendrocyte lineage (Olig2, arrows in o–q) or cerebellar interneurons (Parvalbumin, arrows in r–t and Pax2, arrows in u–w). Imunohistochemical stainings were performed on 4 µm FFPE sections. White lines show perimeter of Purkinje cell layer. Scale bar in panel a equates to 500 µm, bar in panel b to 50 µm. Scale bars in panels c - w equate to 15 μm.

Figure 2

Undifferentiated Sox2⁺ cells express stemness marker Nestin and show reactive properties. Undifferentiated Sox2⁺ cells are characterized by Nestin expression, detected by Nestin-dependent GFP expression and the lack of Blbp expression (a–c). Additionally, Sox2⁺ Nestin⁺ cells show proliferative capacity as shown by Ki67 labeling (d–f). Sox2⁺ S100⁻ cells were sparsely distributed in the PCL of control animals (g–i) when compared to animals that received a week of physical training (arrows in j–l). Training of mice on the RotaRod system lead to a significant increase in the number of Sox2⁺ S100⁻ cells within the PCL as compared to controls (m – Mann-Whitney-U test; SEM). Immunohistochemical staining in panels a – f was performed on 4 µm FFPE sections. Staining shown in panels g – l was performed on 50 µm free-floating vibratome sections. White lines show perimeter of Purkinje cell layer. Scale bars equate to 50 µm in panels a – f and 100 µm in panels g – l.

Figure 3

Reactivity of SOX2⁺ S100⁻ cells in the human cerebellum. In human cerebellar tissue (a) we found SOX2⁺ S100⁻ cells intermingled with the Bergmann glia of the Purkinje cell layer (arrows in b–d). Upon damage to the cerebellum by cerebellar infarcts, we observed reactive gliosis with massive expansion of the Bergmann glia (e) as well as expansion of SOX2⁺ S100⁻ cells (f–i, standard t-testing; SEM). Immunohistochemical staining was performed on 4 µm FFPE sections. Scale bars in panels a and e equate to 200 µm, scale bars for panels b – h equate to 50 µm.

Figure 4

Neurogenesis from Sox2⁺ cells in the adult cerebellar cortex. Combined BrdU birth-dating and fate-mapping experiments show RFP⁺ progeny from Sox2⁺ cells (a) that had incorporated BrdU during the observation period (b) and had also acquired neuronal differentiation as shown by staining for the granule cell marker NeuN (c). Nuclear DAPI staining is shown as a reference in panel d. A maximum intensity projection is shown in panel e with orthogonal views of x-z and y-z planes. The images have been taken after a treatment course with Tamoxifen and BrdU according to the scheme in Supplementary Figure 2. An increase in neurogenesis from Sox2⁺ cells could be achieved by physical activity. Mice that were trained on the RotaRod showed significantly more new-born granule neurons in the internal granule cell layer (IGL), when compared to control animals (f,g – Mann-Whitney-U test; SEM). RotaRod data were obtained after a Tamoxifen/BrdU treatment according to the scheme in Supplementary Figure 2. Possible integration of progeny from Sox2⁺ progenitors is shown by positive staining for the activity regulated transcription factor cFos (h–l). Immunohistochemical staining was performed on 50 µm free-floating vibratome sections. Scale bars for panels a – d and h – k equate to 40 µm, scale bars in panels e and l equate to 10 µm.